The present invention relates to a novel bioreactor design for expressing and separating a biological product from other components in a bioreaction broth, which combines the step of expressing and separating within the bioreactor by binding the biological product with a resin within a bioreactor, discarding the nutrient medium and eluting the biological product as a concentrated solution; this allows elimination at least two steps in the separation and purification of biological products—filtration or centrifugation to remove cell culture and ultrafiltration for volume reduction—and possibly three steps, including loading of biological products on the purification columns.
For products which are expressed as inclusion bodies, the instant invention allows cell lysis, inclusion body solubilization and protein refolding within the bioreactor.
The instant invention significantly reduces the process time and cost while enhancing the yield by reducing degradation of biological products during manufacturing; additional benefits of the instant invention include avoiding perfusion process and reducing toxicity of the expressed biological products to cell culture. No such invention exists in the prior art of bioreactors.
Downstream processing involves steps for cleaning up crude biological products to yield high purity products. Traditionally, these steps involve using chromatography columns packed with highly specialized resins to capture and purify the desired biological products by the process of elution. With an exponential rise in the number of biological products being developed and marketed, there have been remarkable developments in the field of downstream processing; these have however not caught up with the developments in the upstream processing. A few years ago, an yield of 0.25 G of biological product per liter expressed by CHO cells was considered very high; today, we are hovering yields around 10 G/L making it possible to accumulate a very large quantity of biological products, particularly as the sizes of bioreactors have increased to thousands of liters. There are three steps that connect the upstream and downstream processing. First, the culture media must be filtered using fine filters (e.g., 0.22 microns) to remove cells (CHO cells have average size of 5 microns). This step utilizes an array of filters since the cells are likely to choke the filter surface easily and also require installing vessels that would receive the filtrate. This requires vessels of thousands of liters of capacity to match the size of the bioreactors. The next step is the reduction of the volume of filtrate since it is not possible to load such large volumes on columns that have limited flow rate. This is the stage where most often a cross-flow type filtration is used, again with a large bank of filters to complete the concentration process as quickly as possible. The mechanism of cross flow filtration place severe pressure on the solution and causes breakdown and precipitation of biological products resulting in losses of generally 10-20% at this stage. Both of these processes take a very long time and during this processing it is not possible to keep the biological product solution at a lower temperature resulting in the degradation of biological product as well. The third step is to load the concentrated solution in a chromatography column containing a binding media, a specific resin with affinity for the target biological product. Even though the volume of liquid has been reduced considerably at this stage, the loading steps, nevertheless, takes substantial time to complete the loading.
The time and cost-consuming steps of filtration, chromatography and purification slow down the manufacturing process and add substantial capital cost requirement to establish cGMP-grade manufacturing operations.
Bioreactors used in the upstream processing are vessels that allow growth of cell culture to express biological products and for reasons historic and traditional, a clear demarcation line exists between the expression of biological product and its purification. For this reason, no innovations have been made to add additional functions to the design of bioreactors while they do provide a large investment in a vessel that could possibly have multiple uses.
There is a large unmet need to stream line the entire process of biological manufacturing of products where the cost of manufacturing can be reduced substantially but combining several traditional steps in a single vessel, the bioreactor.
The instant invention discloses an innovative bioreactor design that accomplishes this goal and is applicable universally to all types of bioreaction applications.